Shinji Matsumoto

Wnt5a: its signalling, functions and implication in diseases

Wnt5a is a representative ligand that activates the β‐catenin‐independent pathways. Because the β‐catenin‐independent pathway includes multiple signalling cascades in addition to the planar cell polarity and Ca2+ pathway, Wnt5a regulates a variety of cellular functions, such as proliferation, differentiation, migration, adhesion and polarity. Consistent with the multiple functions of Wnt5a signalling, Wnt5a knockout mice show various phenotypes, including an inability to extend the embryonic anterior–posterior and proximal–distal axes in outgrowth tissues. Thus, many important roles of Wnt5a in developmental processes have been demonstrated. Moreover, recent reports suggest that the postnatal abnormalities in the Wnt5a signalling are involved in various diseases, such as cancer, inflammatory diseases and metabolic disorders. Therefore, Wnt5a and its signalling pathways could be important targets for the diagnosis and therapy for human diseases.

New insights into the mechanism of Wnt signaling pathway activation.

Wnts compromise a large family of secreted, hydrophobic glycoproteins that control a variety of developmental and adult processes in all metazoan organisms. Recent advances in the Wnt-signal studies have revealed that distinct Wnts activate multiple intracellular cascades that regulate cellular proliferation, differentiation, migration, and polarity. Although the mechanism by which Wnts regulate different pathways selectively remains to be clarified, evidence has accumulated that in addition to the formation of ligand-receptor pairs, phosphorylation of receptors, receptor-mediated endocytosis, acidification, and the presence of cofactors, such as heparan sulfate proteoglycans, are also involved in the activation of specific Wnt pathways. Here, we review the mechanism of activation in Wnt signaling initiated on the cell-surface membrane. In addition, the mechanisms for fine-tuning by cross talk between Wnt and other signaling are also discussed.

Binding of APC and dishevelled mediates Wnt5a‐regulated focal adhesion dynamics in migrating cells

Wnt5a is a representative ligand that activates the Wnt/β‐catenin‐independent pathway, resulting in the regulation of cell adhesion, migration, and polarity, but its molecular mechanism is poorly understood. This report shows that Dishevelled (Dvl) binds to adenomatous polyposis coli (APC) gene product, and this binding is enhanced by Wnt5a. Dvl was involved in the stabilization of the plus end dynamics of microtubules as well as APC. Frizzled2 (Fz2) was present with Wnt5a at the leading edge of migrating cells and formed a complex with APC through Dvl. Fz2 also interacted with integrins at the leading edge, and Dvl and APC associated with and activated focal adhesion kinase and paxillin. The binding of APC to Dvl enhanced the localization of paxillin to the leading edge and was involved in Wnt5a‐dependent focal adhesion turnover. Furthermore, this new Wnt5a signalling pathway was important for the epithelial morphogenesis in the three‐dimensional culture. These results suggest that the functional and physical interaction of Dvl and APC is involved in Wnt5a/Fz2‐dependent focal adhesion dynamics during cell migration and epithelial morphogenesis.

Localization of glypican-4 in different membrane microdomains is involved in the regulation of Wnt signaling

Glypicans are members of the heparan sulfate proteoglycans (HSPGs) and are involved in various growth factor signaling mechanisms. Although HSPGs affect the β-catenin-dependent and -independent pathways of Wnt signaling, how they regulate distinct Wnt pathways is not clear. It has been suggested that the β-catenin-dependent pathway is initiated through receptor endocytosis in lipid raft microdomains and the independent pathway is activated through receptor endocytosis in non-lipid raft microdomains. Here, evidence is presented that glypican-4 (GPC4) is localized to both membrane microdomains and that the localization affects its ability to regulate distinct Wnt pathways. GPC4 bound to Wnt3a and Wnt5a, which activate the β-catenin-dependent and -independent pathways, respectively, and colocalized with Wnts on the cell surface. LRP6, one of Wnt3a coreceptors, was present in lipid raft microdomains, whereas Ror2, one of Wnt5a coreceptors, was localized to non-lipid raft microdomains. Expression of GPC4 enhanced the Wnt3a-dependent β-catenin pathway and the Wnt5a-dependent β-catenin-independent pathway, and knockdown of GPC4 suppressed both pathways. A GPC4 mutant that was localized to only non-lipid raft microdomains inhibited the β-catenin-dependent pathway but enhanced the β-catenin-independent pathway. These results suggest that GPC4 concentrates Wnt3a and Wnt5a to the vicinity of their specific receptors in different membrane microdomains, thereby regulating distinct Wnt signaling.

An Anti-Wnt5a Antibody Suppresses Metastasis of Gastric Cancer Cells In Vivo by Inhibiting Receptor-Mediated Endocytosis

Wnt5a is a representative ligand that activates the β-catenin–independent pathway in Wnt signaling. It was reported that the expression of Wnt5a in human gastric cancer is associated with aggressiveness and poor prognosis and that knockdown of Wnt5a reduces the ability of gastric cancer cells to metastasize in nude mice. Therefore, Wnt5a and its signaling pathway might be important targets for the therapy of gastric cancer. The aim of this study was to examine whether an anti-Wnt5a antibody affects metastasis of gastric cancer cells. One anti-Wnt5a polyclonal antibody (pAb5a-5) inhibited migration and invasion activities in vitro of gastric cancer cells with a high expression level of Wnt5a. Previously, it was shown that Wnt5a induces the internalization of receptors, which is required for Wnt5a-dependent activation of Rac1. pAb5a-5 inhibited Wnt5a-dependent internalization of receptors, thereby suppressed Wnt5a-dependent activation of Rac1. Laminin γ2 is one of target genes of Wnt5a signaling and Rac1 was involved in its expression. pAb5a-5 also inhibited Wnt5a-dependent expression of laminin γ2. In an experimental liver metastasis assay, gastric cancer cells were introduced into the spleens of nude mice. Laminin γ2 was required for liver metastatic ability of gastric cancer cells in vivo. Furthermore, intraperitoneal injection of pAb5a-5 inhibited the metastatic ability of gastric cancer cells. These results suggest that an anti-Wnt5a antibody was capable of suppressing Wnt5a-dependent internalization of receptors, resulting in the prevention of metastasis of gastric cancer cells by inhibiting the activation of Rac1 and the expression of laminin γ2. Mol Cancer Ther; 11(2); 298–307. ©2011 AACR.

The Dishevelled-associating protein Daple controls the non-canonical Wnt/Rac pathway and cell motility

Dishevelled is the common mediator of canonical and non-canonical Wnt signalling pathways, which are important for embryonic development, tissue maintenance and cancer progression. In the non-canonical Wnt signalling pathway, the Rho family of small GTPases acting downstream of Dishevelled has essential roles in cell migration. The mechanisms by which the non-canonical Wnt signalling pathway regulates Rac activation remain unknown. Here we show that Daple (Dishevelled-associating protein with a high frequency of leucine residues) regulates Wnt5a-mediated activation of Rac and formation of lamellipodia through interaction with Dishevelled. Daple increases the association of Dishevelled with an isoform of atypical protein kinase C, consequently promoting Rac activation. Accordingly, Daple deficiency impairs migration of fibroblasts and epithelial cells during wound healing in vivo. These findings indicate that Daple interacts with Dishevelled to direct the Dishevelled/protein kinase λ protein complex to activate Rac, which in turn mediates the non-canonical Wnt signalling pathway required for cell migration.

The Wnt5a-Ror2 axis promotes the signaling circuit between interleukin-12 and interferon-γ in colitis

Wnt5a, which regulates various cellular functions in Wnt signaling, is involved in inflammatory responses, however the mechanism is not well understood. We examined the role of Wnt5a signaling in intestinal immunity using conditional knockout mice for Wnt5a and its receptor Ror2. Removing Wnt5a or Ror2 in adult mice suppressed dextran sodium sulfate (DSS)-induced colitis. It also attenuated the DSS-dependent increase in inflammatory cytokine production and decreased interferon-γ (IFN-γ)-producing CD4+ Th1 cell numbers in the colon. Wnt5a was highly expressed in stromal fibroblasts in ulcerative lesions in the DSS-treated mice and inflammatory bowel disease patients. Dendritic cells (DCs) isolated from the colon of Wnt5a and Ror2 deficient mice reduced the ability to differentiate naïve CD4+ T cells to IFN-γ-producing CD4+ Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-γ, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-γ signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis.

The WNT/MYB pathway suppresses KIT expression to control the timing of salivary proacinar differentiation and duct formation

Growth factor signaling is involved in the development of various organs, but how signaling regulates organ morphogenesis and differentiation in a coordinated manner remains to be clarified. Here, we show how WNT signaling controls epithelial morphogenetic changes and differentiation using the salivary gland as a model. Experiments using genetically manipulated mice and organ cultures revealed that WNT signaling at an early stage (E12-E15) of submandibular salivary gland (SMG) development inhibits end bud morphogenesis and differentiation into proacini by suppressing Kit expression through the upregulation of the transcription factor MYB, and concomitantly increasing the expression of distal progenitor markers. In addition, WNT signaling at the early stage of SMG development promoted end bud cell proliferation, leading to duct formation. WNT signaling reduction at a late stage (E16-E18) of SMG development promoted end bud maturation and suppressed duct formation. Thus, WNT signaling controls the timing of SMG organogenesis by keeping end bud cells in an undifferentiated bipotent state. Highlighted article: During branching morphogenesis of the salivary gland, mesenchymal WNT signals promote duct formation and inhibit KIT-mediated proacinar differentiation.

Prickle1 promotes focal adhesion disassembly in cooperation with the CLASP–LL5β complex in migrating cells

ABSTRACT Prickle is known to be involved in planar cell polarity, including convergent extension and cell migration; however, the detailed mechanism by which Prickle regulates cellular functions is not well understood. Here, we show that Prickle1 regulates front-rear polarization and migration of gastric cancer MKN1 cells. Prickle1 preferentially accumulated at the cell retraction site in close proximity to paxillin at focal adhesions. Prickle1 dynamics correlated with those of paxillin during focal adhesion disassembly. Furthermore, Prickle1 was required for focal adhesion disassembly. CLASPs (of which there are two isoforms, CLASP1 and CLASP2, in mammals) and LL5β (also known as PHLDB2) have been reported to form a complex at cell edges and to control microtubule-dependent focal adhesion disassembly. Prickle1 was associated with CLASPs and LL5β, and was required for the LL5β-dependent accumulation of CLASPs at the cell edge. Knockdown of CLASPs and LL5β suppressed Prickle1-dependent cell polarization and migration. Prickle1 localized to the membrane through its farnesyl moiety, and the membrane localization was necessary for Prickle1 to regulate migration, to bind to CLASPs and LL5β, and to promote microtubule targeting of focal adhesions. Taken together, these results suggest that Prickle1 promotes focal adhesion disassembly during the retraction processes of cell polarization and migration. Highlighted Article: Prickle localizes adjacent to focal adhesions and regulates microtubule targeting to focal adhesions in cooperation with CLASPs and LL5β, thereby promoting focal adhesion disassembly to stimulate cell migration.

Regulation of focal adhesion dynamics by Wnt5a signaling.

Wnt5a is a representative ligand that activates the β-catenin-independent pathway of Wnt signaling in mammals. This pathway might be related to planar cell polarity signaling in Drosophila. Because reliable biochemical assays to measure Wnt5a pathway activity have not yet been established, we examined whether Wnt5a signaling stimulates focal adhesion turnover in migrating cells using live immunofluorescence imaging and immunocytochemical analysis. These assays demonstrated that the Wnt5a pathway cooperates with integrin signaling to regulate cell migration and adhesion through focal adhesion dynamics.